Prostate Cancer PA0 Probasin Gene PA0 SV40 T Antigen PA0 Transgenic Animal Models
Prostate cancer will likely claim the lives of 35,000 men in the United States this year alone and some 200,000 more men will be diagnosed with the disease (Silverger, Boring & Squires, 1990). However, progress toward understanding the biology of prostate cancer and the development of new therapies for this disease has been slowed, in part, by the need for new in vivo model systems that adequately reproduce the spectrum of benign, latent, aggressive and metastatic forms of the human disease.
Prostate cancer is a disease quite unique to man. Although naturally occurring prostatic disease has been reported in some canine (Berry, Coffey, & Ewing, 1984) and rodent (Noble, 1977; Pollard, 1973; Pollard & Luckert, 1987; Shain, McCullough, Nitchuk, & Boesel, 1977; Shain, McCullough, & Segaloff, 1975) species, these animals have not provided the appropriate models to adequately study the molecular mechanisms related to the early development and progression of human prostate cancer. To this end, we initiated a research program to establish a transgenic animal model for prostate cancer using a prostate-specific transgene expression system that has been developed based on the regulatory elements of the rat probasin (rPB) gene.
The rPB gene encodes an androgen- and zinc-regulated protein specific to the dorsolateral epithelium (Dodd, Sheppard & Matusik, 1983; Matusik, Kreis, McNicol, Sweetland, Mullin, Fleming, et al., 1986; Sweetland, Sheppard, Dodd & Matusik, 1988). Isolation of the rPB gene has facilitated identification of cis-acting androgen response regions within the 5' flanking region (Rennie, Bruchovsky, Leco, Sheppard, McQueen, cheng, et al., 1993). More recently, the ability of the prostate specific rPB gene promoter to target heterologous genes specifically to the prostate in transgenic mice has been demonstrated (Greenber, DeMayo, Sheppard, Barrios, Lebovitz, Finegold, et al., 1994). In these studies, an expression cassette carrying 426 base pairs (bp) of the rPB gene promoter and 28 bp of 5' untranslated region (5'UT) was found to be sufficient to target expression of a bacterial chloramphenicol acetyl transferase (CAT) reporter gene specifically to the prostatic epithelium. These studies demonstrated that the minimal rat probasin promoter was specifically regulated by androgens in vivo with the ability to target developmentally- and hormonally- regulated expression of a heterologous gene specifically to the prostate in transgenic mice.
The latter studies, showing prostate-specific targeting of heterologous gene expression products is described in the aforementioned U.S. Ser. No. 08/351,365 and published in the corresponding WO 94/03594, the disclosures of which are incorporated herein by reference.
The SV40 early region-tumor antigens (Tag) have the ability to induce transformation in vivo (Brinstar, Chen, Messing, Van Dyke, Levine, & Palmiter, 1984). The SV40 large T antigen acts as an oncoprotein through interactions with the retinoblastoma(Rb) (DeCaprio, Ludlow, Figge, Shew, Huang, Lee, et al., 1988) and p53 (Lane & Crawford, 1979; Linzer & Levine, 1979) tumor suppressor gene products. The small t antigen interacts with a protein phosphatase (Pallas, Shahrik, Martin, Jaspers, Miller, Brautigan, et al., 1990) presumably to regulate activity of the mitogen activated protein kinase activation pathway and the AP-1transcription factor activity (Frost, Alberts, Sontag, Guan, Mumby, & Feramisco, 1994). The SV40 early region tumor antigens have been used successfully in transgenic mice to induce a transformed state in a variety of systems from pancreas (Hanahan, 1985), mammary gland (Tzeng, Guhl, Graessmann, & Graessmann, 1993), to the ductal epithelium of lung and kidney (Choi, Lee & Ross, 1988) and others (see (Adams & Cory, 1991) for review). Since the loss of wild-type p53 and Rb have been implicated in development and progression of prostate cancer (Bookstein, Rio, Madreperla, Hong, Allred, Grizzle, et al., 1990; Cooke, Quarmby, Mickey, Isaacs, & French, 1988; Isaacs, Carter, & Ewing, 1991; Rubin, Hallahan, Ashman, Brachman, Beckett, Virudachalam, et al., 1991), we hypothesized that directly expressing SV40 tumor antigens in the prostate epithelium of transgenic mice might provide a new mouse model for the development and progression of prostate cancer.
Previous efforts to establish experimental animal models of prostate cancer have explored other primate systems (Habenicht, el-Etreby, Lewis, Ghoniem, & Roberts, 1989), or have focussed primarily on the effects of sex hormones that displays a very low frequency of spontaneous prostatic disease. For example, rats treated with sex hormones (Noble, 1977) developed adenocarcinoma of the prostate, as did rats treated with methylnitrosourea (Pollard & Luckert, 1987), testosterone and cyproterone (Pollard, Luckert & Snyder, 1989) or methylnitrosourea, dimethylbenzanthracene, or dimethylaminobiphenyl after sequential treatment with cyproterone acetate and testosterone propionate (Bosland & Prinsen, 1990; Bosland, Prinsen, Dirksen, & Spit, 1990). In addition, prostate tumors arise following cadmium (Waalkes, Rehm, Riggs, Bare, Devor, Poirer, et al., 1988) administration to WistarCrl:(WI)BR! rats and estradiol and dimethylaminobiphenyl (Shirai, Fukushima, Ikawa, & Ito, 1986) administration to F344 rats. Unfortunately, these protocols induce highly variable primary tumors and are not amenable to studying the molecular events involved in the progression of latent locally defined disease to adenocarcinoma and metastasis. Similarly, efforts to develop in vitro models for prostate cancer have produced the Dunning (Bogden, Taylor, Moreau, & Coy, 1990) (Cooke, et al., 1988), PC-3 (Rubin, et al., 1991) and PC-82 (van & Schroder, 1988) cell lines that are capable of inducing xenograft tumors. More recently, new LnCaP sublines have been developed that are androgen-independent (Thalman, Anezinis, Chang, Zhau, Kim, Hopwood, et al., 1994). However, these systems do not facilitate characterization of the earliest events in the progression of prostate cancer and established tumor cell lines may have been selected either for a number of genetic alterations not found in the primary tumors or acquired mutations in vitro (Carroll, Voeller, Sugars, & Gelmann, 1993; Rubin, et al., 1991). To this end, in vivo gene transfer studies were pursued recently in an attempt to develop a new generation of prostate cancer research models.
Retrovirus mediated gene transfer has been used to develop a mouse prostate reconstitution model (MPR). In this system, the Ha-ras and c-myc genes are first introduced into the fetal urogenital sinus and a reconstituted organ is then transplanted under the renal capsule of host mice leading to the rapid development of adenocarcinoma (Thompson, Southgate, Kitchener, & Land, 1989). MPR has been used previously to demonstrate that co-operativity between Ha-ras and c-myc leads to the transformation of reconstituted prostates (Thompson, Kadmon, Timme, Merz, Egawa, Krebs, et al., 1991; Thompson, Timme, Kadmon, Park, Egawa, & Yoshida, 1993a; Thompson, Truong, Timme, Kadmon, McCune, Flanders, et al., 1993b; Thompson, et al., 1989), and that dietary fenretinide can reduce the incidence of these tumors (Slawin, Kadmon, Park, Scardino, Anzano, Sporn, et al., 1993). Furthermore, MPR studies have demonstrated that TGF.beta.1 may be a marker in the progression of prostate disease from benign to malignant status (Thompson, et al., 1993b), and more recently, that p53 inactivation is related to increase tumorigenesis, and metastasis (Timme, Park, Ren, Eastham, Baley, Kadmon, et al., 1994).
Previous attempts by other investigators to establish transgenic targeting systems based on the regulatory elements of cellular or viral genes known to be expressed in the prostate have met only with limited success.
Studies in vivo demonstrate that C3(1) is induced 2-to-4 fold transcriptional by androgens (Parker et al, 1988; Parker and Needham, 1985), causing the authors to suggest post-transcriptional regulation (page and Parker, 1982).
When a fragment of the rat C3(1) gene was fused to a heterologous .beta.-gal reporter gene, the spatial pattern of C3(1)-.beta.gal fusion transgene expression was not found to be uniform (Buttyan & Slawin, 1993). Mice carrying a C3(1)-Tag transgene have recently been generated (Maroulakou, Anver, Garrett, & Green, 1994). In the two lines of C3(1)-Tag mice that have been propagated, the males develop prostatic hyperplasia and adenocarcinoma while the female mice develop mammary carcinoma. In general, the majority of male mice were observed to develop prostate adenocarcinoma by 8 months of age. Interestingly, a number of phenotypic abnormalities were also observed in the founder mice with only two animals surviving longer than 20 weeks. The founders displayed osteosarcomas, proliferative lesions of the thyroid, salivary glands and nasal epithelium as well as an unusual form of chondrodysplasia. Therefore, the C3(1)-Tag construct used in these studies appears to lack regulatory regions required to limit expression exclusively to the prostate. In addition, the C3(1) transgene is responsive to both male and female sex hormones. In another case, mice carrying a truncated mouse mammary tumor virus long terminal repeat (MMTV-LTR)-int-2 cDNA construct were originally reported to exhibit a phenotype consistent with benign prostatic hyperplasia (Muller, Lee, Dickson, Peters, Pattengale, & Leder, 1990; Tutrone, Ball, Ornitz, Leder, & Richie, 1993). However, further histological evaluation has revealed the pathology actually occurs in the ampullary gland (G. Cunha, unpublished observations). When a fragment of the promoter for human prostate specific antigen (PSA) was used to target an activated Ha-ras oncogene to the prostate in transgenic mice, the mice developed salivary gland tumors with no apparent pathology in prostate gland (Schaffner, Barrios, Shaker, Rajagopalan, Huang, Tindall, et al., 1994). It is interesting to note that the promoter for the PSA gene, a member of the kallikrein gene family (Riegman, Vlietstra, van, Romijn, & Trapman, 1989), directed expression in salivary gland in transgenic mice since similar observations were not reported in previous transgenic studies using a kallikrein rKlK1 promoter Tag constructs (Smith, Lechago, Wines, MacDonald, & Hammer, 1992).